| Objective:Damp-Heat syndrome of spleen-stomach indicates to be affected by the damp-heat pathogen,over-eating hot-fa foods,dampness encumbering the spleen,etc.however,the disease is getting around in Guangdong and Guangxi province.While the syndrome in very diffic?lt to be treat res u 1 t from its complicated variable clinical manifestations and hard to recover.Aiming at etiology and integrating with our prior experimental methods,the animal models based on the composite element of "inner damp-heat,outside damp-heat and chemical agent" was applied.The general survival situation,the body mass(BM),rectal temperature(RT),food and water consumption,histopathological changes of the gastrointestinal mucosa were observed.The expression of HSP70,HIF-la and VEGF were detected by SABC immunohistochemical as well as western-blotting.Pathological changes of gastric,duodenal,colon tissues were observed using H.E.by the light microscope.In the end we want to probe the pathomechanism of damp-heat syndrome of spleen-stomach and Wang-shi-lian-pu-yin,furthermore to provide new evidences on the biology and pathomechanism of damp-heat syndrome of spleen-stomach,at last to improve the overall understanding of damp-heat syndrome of spleen-stomach both the research and clinical facets.Methods:60 male SD rats from SPF grade were raised around three days,and after 12 hour fasting,were randomly divided into 6 groups:normal control group,Damp-Heat of Spleen-Stomach syndrome model group,Omeprazole group,Wang-shi-lian-pu-yin decoction groups of high,medium and low doses.Besides the normal Rats,others were feed with 22 days high-sugar and high-lipid fodder(general fodder+20%hydromel+ grease),22 days chemical agent(5%Sodium Salicylate+ white wine).From the 16th day,rats were put in 7 days artificial heat climate sim?l ation cabin external hot and humid environment(site:dry b?l b temperature 32±0.5?,relative humidity 70±5%,from 9 to 11 am),rats were out of that artificial environment in 23th day,after all,the model of damp-heat syndrome of spleen-stomach was finished.Then Omeprazole group and three WSLPY groups(high,medium and low doses)were given medicine through administration by gavage with 2ml/200g/day for 7 days,the normal control and model group received equal volume of saline once a day.Water drinking and food intake were recorded everyday,the body weight was measured before model making,the fifteenth,twenty second days.The rectal temperature was measured before and after model making,after the treatment.Gastric mucous tissues were received pathohistological examination under light microscope.The expression of HSP70,HIF-la and VEGF proteins were detected by SABC immunohistochemical as well as western-blotting.Statistical analysis of repeated measure ANOVA and One-way ANOVA by SPSS 13.0 software were applied to tackle with the data,P<0.05 represents statistical significance.Res?lts:1.Variation tendency of the general state,food and water consumptionIn the process of the whole model-building,the normal group rats were in great mental state with smooth fur,the activities of a flexible,sensitive response,reg u 1 ar food and water consumption,ordinary bowels.With the time flying of the model-building process,the damp-heat group and treated group rats gradually became sluggishness,lack of move,less food and water intake,withered fur,d?ll reaction and loose fop,l ing stool.While the treated group rats became better mental state,more movements,increased food and water consumption,nearly normal stool.During the model-building the rats in the model group have a decent food and water consumption,which was no significant statistical difference(P>0.05).The food consumption of the medicine treated group rats were rose significantly which was significant statistical difference(P<0.01,P<0.05),otherwise the water consumption had little increased which was no significant statistical difference(P>0.05).2.Variation tendency of the body weight and rectal temperature.In the process of the whole model-building,weight gradually increased in normal group rats and rectal temperature has no significant changes.Compared with normal group,BW grew slowly in model group which was no significant statistical difference(P>0.05),RT remarkable elevated which was significant statistical difference(P<0.01).BW grew little faster in WSLPY groups and Omeprazole group than model group which was no significant statistical difference(P>0.05),While RT went down rapidly(P<0.01)in the WSLPY groups and Omeprazole group.3.Variation of the pathology of gastric mucous tissue in rats with damp-heat syndrome of spleen-stomachHE observed under electron lens dyeing glass displayed:the gastric mucous tissues in the normal group were no characteristic differences which is structure intact;but of the model group rats,which were seen in gastric mucosa tissue to tissue congestion,accompanied by edema and large amounts of neutral grain and eosinophils in gastric mucosa lamina propria and submucosa inflammatory infiltrates.Compared to the model group,the structure of rat gastric mucosa were clear well Lamina propria were scattered inflammatory cells invasion in Omeprazole group and WSLPY groups.4.Variation of the HSP70 protein expression in rats with damp-heat syndrome of spleen-stomachImmunohistochemical image analysis res?lts showed up:There exists over expression of HSP70 in model group with dispersed distribution,Cell cytoplasm were dyed buffy,dark shading,which represents strong positive expression.The OD value remarkable elevated which was significant statistical difference(P<0.01).Compare with the damp-heat syndrome of spleen-stomach group,The OD of WSLPY groups show a significant reduction(P<0.01).Omeprazole group's OD has a reduction tend(p<0.05);Compared with that,low-dose WSLPY group's OD has a reduction but of no significant difference(P>0.05),but the middle and high-dose group's OD has a significant reduction which was significant statistical difference(P<0.01).WB showed:The expression of HSP70 in the model group,OME group,WSLPY groups higher than in the normal group,which has significant difference(P<0.01)HSP 70 in OME group,WSLPY groups are lower than DHSS group significantly(P<0.05).Compare with OME group,WSLPYs lower significantly(P<0.01);the WSLPY groups reflected no remarkable statistical difference.5.Variation of the HIF-1? protein expression in rats with damp-heat syndrome of spleen-stomach.IHC analysis res?lts showed up:There exists over expression of HIF-1? in model group with dispersed distribution,Cell cytoplasm were dyed buffy,dark shading,which represents strong positive expression.The OD value remarkable elevated which was significant statistical difference(P<0.01).The WSLPY groups and OME group showed a moderate expression,OD decreased significantly(p<0.01);Compared to the OME group,The WSLPY middle and high-dose groups' OD decreased significantly(p<0.05);The WSLPY low-dose group has a weak expression,OD decreased of no significant(p>0.05).WB show:The expression of HIF-1? in the model group,OME goup,WSLPY groups higher than in the normal group,which has significant difference(P<0.01);HIF-1? in OME group,WSLPY groups are lower than normal group significantly(P<0.01).Compare with OME group,WSLPY groups lower significantly(P<0.01);Among the WSLPYs,there is no significant difference,but the best one is the middle-dose group.6.Variation of the VEGF protein expression in rats with damp-heat syndrome of spleen-stomachIHC analysis res?l ts showed up:There exists over expression of VEGF in model group with dispersed distribution,Cell cytoplasm were dyed buffy,dark shading,which represents strong positive expression.The OD value remarkable elevated which was significant statistical difference(P<0.01).Compare with the model group,The WSLPY groups show a moderate expression,OD decreased significantly(p<0.01);OME group's reduction of no significant(p>0.05),the OD of every dose of The WSLPY groups all lower than OME group which was significant statistical difference(P<0.05,P<0.01).WB showed:The expression of VEGF in the model group,OME goup higher than in the normal group,which has significant difference(P<0.01);Compare with WSLPY groups,there is no significant difference;OME group and WSLPYs reduce significant difference(P<0.01);Compared with OME group,WLPY groups have a lower expression,which has significant difference(P<0.05);There is no significant difference among the WSLP groups(P<0.05).Conclusion:1.The animal models based on the composite element of "inner damp-heat,outside damp-heat and chemical agent" is copied successf?lly.The model group rats appears sluggishness,lack of move,less food and water intake,withered fur,d?ll reaction and loose fo?l ing stool.The res?It is in line with the frequent disease of damp-heat syndrome of spleen-stomach.2.Remarkable elevated of HSP70,HIF-la and VEGF reflects that mutual manip?l ation among with the proteins,which is the vital component of the pathogenesis of damp-heat syndrome of spleen-stomach.Also there is a hint that when the body under the stress state of in the damp-heat circumstance stim?l ation which reflects "battle of health and illness" which is the basic theory of TCM.3.Wang-sh-lian-pu-yin can cure the syndrome of damp-heat spleen-stomach of the rats by clearing heat and expelling damp,which may through the mutual manip?l ation of the HSP70,HIF-1? and VEGF.4.Imunohistochemical and immune imprinting res?l ts suggest that there exist over expression of HSP70,HIF-1 a and VEGF,meanwhile HE show Inflammatory damage and bleeding,which point out Wang-shi-lian-pu-yin provides the therapeutic action by means of the thermal stress in the body of the inflammatory response,to maintain stable internal environment,change in the reaction of inflammation,improve gastric mucosa microcirc?l ation,promote restoration of the gastric mucosa damage.Furthermore,Wan-shi-lian-pu-yin may provide a better therapeutic effect than omeprazole. |
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